Designation: E 925 – 02

Standard Practice for

Monitoring the Calibration of Ultraviolet-Visible
Spectrophotometers whose Spectral Slit Width does not
Exceed 2 nm1
This standard is issued under the fixed designation E 925; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.

INTRODUCTION

In the application of spectrophotometric methods of analysis it is the responsibility of the analyst

to verify and validate that the instrument is functioning properly and is capable of providing
acceptable analytical results. It is preferable that the verification of instrument performance be
accomplished through the use of reference materials whose properties have been accurately
determined. Such materials are readily available, and their use in the tests and measurements described
in this practice is satisfactory for evaluating the performance of spectrophotometers whose spectral slit
width does not exceed the value for which the intrinsic or certified properties are valid. A compromise
maximum permissible spectral slit width of 2 nm is recommended for the reference materials and error
tolerances recommended here.
This practice covers some of the essential instrumental parameters that should be evaluated to
ensure the acceptability of the analytical data routinely obtained on the instrument. These parameters
include the accuracy of the wavelength and absorbance scales and stray radiant power levels.
The accuracy of the wavelength scale in both the UV and visible regions is determined using the
sharp absorption bands of a holmium oxide glass or solution filter. The absorbance scale accuracy in
the UV region (235–350 nm) is determined using acidic solutions of potassium dichromate. In the
visible region (440–635 nm) the absorbance accuracy is determined using individually certified neutral
density glass filters. The use of these reference materials provides a valid and relatively simple means
to test the errors in the wavelength and absorbance scales of small spectral slit width spectrophotom-
eters in the spectral ranges indicated. A simplified version of the opaque filter method is provided as
a test for excessive stray radiant energy.

1. Scope specifications for wavelength and absorbance accuracy do not

1.1 This practice covers the parameters of spectrophotomet- exceed the performance tolerances employed here. This prac-
ric performance that are critical for testing the adequacy of tice employs an illustrative tolerance of 61 % relative for the
instrumentation for most routine tests and methods2 within the error of the absorbance scale over the range of 0.2 to 2.0, and
wavelength range of 200 to 700 nm and the absorbance range of 61.0 nm for the error of the wavelength scale. A suggested
of 0 to 2. The recommended tests provide a measurement of the maximum stray radiant power ratio of 4 3 10-4 yields <1 %
important parameters controlling results in spectrophotometric absorbance bias at an absorbance of 2. These tolerances are
methods, but it is specifically not to be inferred that all factors chosen to be compatible with many chemical applications
in instrument performance are measured. while comfortably exceeding the uncertainty of the certified
1.2 This practice may be used as a significant test of the values for the reference materials and typical manufacturer’s
performance of instruments for which the spectral slit width specifications for error in the wavelength and absorbance
does not exceed 2 nm and for which the manufacturer’s scales of the instrument under test. The user is encouraged to
develop and use tolerance values more appropriate to the
requirements of the end use application. This procedure is
1
This practice is under the jurisdiction of ASTM Committee E13 on Molecular designed to verify quantitative performance on an ongoing
Spectroscopy and is the direct responsibility of Subcommittee E13.01 on Ultraviolet
and Visible Spectroscopy. basis and to compare one instrument’s performance with that
Current edition approved March 10, 2002. Published May 2002. Originally of other similar units. Refer to Practice E 275 to extensively
published as E 925 – 83. Last previous edition D 925 – 83 (1994)e1. evaluate the performance of an instrument.
2
Routine tests are defined as those in which absorbance data obtained on a
sample are compared to those of a standard sample preparation.
1.3 This practice should be performed on a periodic basis,

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.

1
 E 925
the frequency of which depends on the physical environment ing data on a periodic basis, forming a base from which any
within which the instrumentation is used. Thus, units handled changes in the performance of the instrument will be evident.
roughly or used under adverse conditions (exposed to dust,
chemical vapors, vibrations, or combinations thereof) should 5. Reference to this Calibration-Monitoring Procedure
be tested more frequently than those not exposed to such 5.1 Reference to this practice in any spectrophotometric
conditions. This practice should also be performed after any calibration-monitoring scheme shall constitute due notification
significant repairs are made on a unit, such as those involving that the adequacy of the spectrophotometer performance has
the optics, detector, or radiant energy source. been evaluated by means of this practice. Performance is
1.4 This standard does not purport to address all of the considered to be adequate when the data obtained are within
safety concerns, if any, associated with its use. It is the the stated tolerances from the true values.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica- 6. Instrument Operation
bility of regulatory limitations prior to use. 6.1 In obtaining spectrophotometric calibration data the
analyst must select the proper instrumental operating condi-
2. Referenced Documents tions to realize satisfactory instrument performance. Operating
2.1 ASTM Standards: conditions for individual instruments are best obtained from
E 131 Terminology Relating to Molecular Spectroscopy3 the manufacturer’s literature because of variations in instru-
E 169 Practices for General Techniques of Ultraviolet/ ment design.
Visible Quantitative Analysis3 6.2 When using reference materials, all the components of
E 275 Practice for Describing and Measuring Performance the spectrophotometer must be functioning properly. In addi-
of Ultraviolet, Visible, and Near Infrared Spectrophotom- tion, the temperature of the specimen compartment should be
eters3 between 20 and 25°C. Matched solution cells should be used
E 387 Test Method for Estimating Stray Radiant Power for calibration purposes.
Ratio of Spectrophotometers by the Opaque Filter Method3 6.3 Each of the above factors in instrument operation is
E 1866 Guide for Establishing Spectrophotometer Perfor- important in the determination of wavelength and absorbance
mance Tests3 accuracy.
2.2 Other Publications:
NBS Special Publication 260-54: Certification and Use of 7. Determination of Wavelength Accuracy in the
Acidic Potassium Dichromate Solutions As An Ultraviolet Ultraviolet and Visible Spectral Regions
Absorbance Standard4 7.1 Discussion—The holmium oxide glass filter (1, 2)5 or
NBS Special Publication 260-102: Holmium Oxide Solu- solution standard (NBS Special Publication 260-102) may be
tion Wavelength Standard from 240 to 640 nm—SRM used for evaluating wavelength accuracy. The glass and solu-
20344 tion standards are both widely available commercially in the
NIST Special Publication 260-116: Glass Filters as a Stan- cuvette format (a cuvette-shaped metal holder is used for the
dard Reference Material for Spectrophotometry— glass), as well as from NIST for high-accuracy needs. Hol-
Selection, Preparation, Certification, and Use of SRM 930 mium glass filters for wavelength calibration checks are
and SRM 19304 available from several distributors of glass color filters and
NIST Special Publication 260-140: Technical Specifications possibly from different glass-manufacturers. A purchaser
for Certification of Spectrophotometric NTRMs4 should require certification by the supplier that the wavelengths
of the absorption bands are within 0.2-nm of the values given
3. Terminology in Reference (2), and reported below. The NIST Calibration
3.1 Definitions: Services program provides individually verified glass wave-
3.1.1 For the definitions of terms used in this practice, refer length standards through Service ID No. 38050C. The appro-
to Terminology E 131. priate solution standard is 4 % (mass fraction) holmium oxide
3.1.2 For a description of the instrumental parameters in 10 % (volume fraction) perchloric acid, contained in a
evaluated in this practice, refer to Practice E 275. 10-mm pathlength cuvette. Band positions for the solution
3.1.3 For a description of quantitative ultraviolet spectro- reference material were determined as locations of the trans-
photometric techniques, refer to Practice E 169. mittance minima of specified bands in the transmittance
spectrum of the material in the original certification of NIST
4. Significance and Use SRM 2034. Absorbance maxima or transmittance minima must
4.1 This practice permits an analyst to compare the perfor- be located within 6 1 nm of the wavelengths given below:
mance of an instrument to the manufacturer’s supplied perfor- Glass FilterA Dilute Acidic SolutionB
mance specifications and to verify its suitability for continued 241.5 nmC 241.1 nm
routine use. It also provides generation of calibration monitor- ... 249.9 nm
279.3 nm 278.1 nm

3
Annual Book of ASTM Standards, Vol 03.06.
4 5
Available from the National Technical Information Service, Port Royal Road, The boldface numbers in parentheses refer to the list of references at the end of
Springfield, VA 22161. this standard.

2
 E 925
Glass FilterA Dilute Acidic SolutionB possible, to verify the nonconformity.
7.2.3 Report the wavelength calibration data in the manner
287.6 nm 287.2 nm
333.8 nm 333.4 nm
of Table 1, given as an example for the holmium oxide glass
... 345.5 nm reference material.
360.8 nm 361.3 nm
385.8 nm 385.7 nm 8. Evaluation of Stray Radiant Power Ratio (SRPR)
418.5 nm 416.3 nm
453.4 nm ...D 8.1 Discussion—A portion of the unwanted stray radiant
459.9 nm 467.8 nm power detected by the photodetector can be measured using the
... 485.3 nm
536.4 nm 536.6 nm
following sharp cut-off solution filters in 1-cm cells:
637.5 nm 640.5 nm
A
Wavelengths taken from Reference (2) for Corning Glass Works Code 3130 Solution Wavelength
glass, superceded by Corning Glass Works Code 3131 glass and Kopp Glass KI, 1.0 g/L in H2O 220 nm
Code 3131 glass, for which the wavelengths are also valid. K2Cr2O7, 0.25 g/L in aqueous 370 nm
B
Wavelengths rounded to 0.1 nm from the results given in NBS SP 260-102 for 0.05 M KOH
a 1-nm spectral slit width.
C
8.1.1 Reagent grade materials should be used for these
May not be usable, depending on the base glass of the filter.
D
Peak omitted because it resolves into a doublet at spectral slit width values
solutions. They are essentially opaque at the indicated wave-
less than 1 nm. lengths; any observed transmittance is equivalent to the effec-
7.1.1 If the observed absorption bands of the holmium oxide tive SRPR.
glass or solution deviate by more than 61 nm from the values 8.1.2 An acceptable level of SRPR depends on the spectral
stated, then corrective service must by performed on the character and absorbance level of the sample under investiga-
instrument by a qualified service person. If the user performs tion. However, an upper limit of 4 3 10-4 is consistent with a
this service, the manufacturer’s recommended procedure worst-case absorbance bias of ~1 % at the upper limit of the
should be followed carefully. absorbance range (0 < A # 2) covered by this practice, and is
7.1.2 The wavelength accuracy is dependent on the spectral suggested in the absence of other criteria.
slit width and thus on the physical slit width. Spectral slit 8.1.3 While the stray radiant power ratio is equivalent to the
widths may be determined from the manufacturer’s specifica- transmittance described previously, it is often more convenient
tions. to make the measurement in the absorbance mode and math-
7.1.3 Computer based peak location algorithms that may be ematically convert absorbance to transmittance.
used to assign absorbance maxima or transmittance minima are 8.1.4 An excessive SRPR usually arises from dust,
discussed in section 7.6 of Guide E 1866. It should be noted scratches, or corrosion on the collimator or disperser, or both.
that peak asymmetries in the holmium oxide reference mate- Qualified personnel should correct this problem. Care should
rials are such that digital filter widths should be smaller than be taken to discriminate between SRPR and light leaks. The
the full-width-half-maximum recommendation of that Guide. latter most often originate in the sample compartment and can
7.1.4 In the absence of drift or slippage in the wavelength be detected by blocking the sample beam alternately at the
drive train, repeatability of the band positions should be on the ports on the source and detector sides of the sample compart-
order of 60.1 nm for a given instrument, especially with the ment. Any difference in the detected signals indicates a light
use of a computer based peak location algorithm. leak.
7.2 Procedure:
7.2.1 Examine the holmium oxide reference material and TABLE 1 UV-VIS Spectrophotometer Wavelength and Stray
Radiant Power Ratio Calibration
remove any surface contamination using a soft brush or lens
Instrument
paper. Measure the temperature of the sample compartment by Date
placing an appropriate sensor into the cell compartment of a Temperature
stabilized instrument and replacing the compartment cover Analyst
Wavelength Calibration: Holmium Oxide Filter
securely. Place the sensor as close as possible to the actual
True Observed Conformance
position that will be occupied by the standard. After a suitable Wavelength Wavelength
Difference
period of time record the temperature reading, remove the (nm) Does Does Not
(nm) (nm)
sensor, and resume normal operations. 241.5 6 1
279.3 6 1
7.2.2 Record the blank absorbance or transmittance (air 287.6 6 1
versus air) spectrum at the desired resolution and at the 333.8 6 1
appropriate wavelength intervals and scan speeds, in order to 360.8 6 1
385.8 6 1
perform any necessary baseline adjustments. The wavelength 418.5 6 1
intervals should be no greater than the spectral slit width used. 453.4 6 1
Acquire the appropriate spectrum of the holmium oxide 459.9 6 1
536.4 6 1
reference material with respect to air and baseline correct if 637.5 6 1
necessary using the blank spectrum. Record the wavelengths of Stray Radiant Power Ratio
the positions of the relevant bands, and compare these values to Wavelength Transmittance
Conforms
Does Not
the expected values. If large discrepancies (>1 nm) exist (nm) or Absorbance Conform
220
between the true and measured wavelengths, repeat the proce- 370
dure at a slower scan speed and smaller spectral slit width, if

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 E 925
8.2 Procedure: transmittance scale maintained by the Analytical Chemistry
8.2.1 Use the visible light source lamp in the 370 nm region Division (ACD) of NIST is supported by NIST for commercial
and the ultraviolet light source lamp in the 220 nm region. participants in the NIST Traceable Reference Materials
8.2.2 Determine the transmittance or absorbance of each (NTRMy) program, or by self assertion for other commercial
solution at the appropriate wavelength using the indicated sources. Traceability of these filters is normally maintained
solvents for reference. through NIST SRM 930 filters with nominal absorbances of
8.2.3 Refer to Standard Test Method E 387 if the dynamic 0.5, 0.7 and 1.0 and SRM 1930 filters with absorbances of 0.3,
range of the readout electronics of the instrument is not 1.5, and 2.0. (A letter series designation for SRM 930 is
adequate for the direct measurement of SRPR as described periodically adjusted without significant effect for this prac-
here. tice.) The wavelengths for which certified absorbance values
8.2.4 In the manner of Table 1, report the transmittance or are reported for individual filters are close to local extrema in
absorbance of these solutions. Note whether the effective stray the nearly-neutral glass to minimize the effect of wavelength
radiant power ratio exceeds the suggested tolerance of 4 3 10-4 error on the measured transmittance:
or the user-defined tolerance. 440.0 nm
465.0 nm
9. Determination of the Absorbance Scale Accuracy in 546.1 nm
the Ultraviolet and Visible Spectral Regions 590.0 nm
9.1 Discussion—The accuracy of the absorbance scale is 635.0 nm
determined using reference materials with known absorbances. Neutral density glass filters are also available from the
The absorbance scale accuracy in the ultraviolet region (235 to National Physical Laboratory (NPL) of the UK and from
350 nm) is determined using acidic solutions of potassium commercial sources asserting traceability to the regular trans-
dichromate as described in National Bureau of Standards mittance scale maintained by NPL.
(NBS) Special Publication 260-54. In the visible region (440 to 9.2.1 These filters have individually certified absorbance
635 nm) the absorbance accuracy is determined using certified values and the precautionary notes stated in the certificate that
neutral density glass filters as described in NIST Special accompanies the filters should be followed. In cases where
Publication 260-116 and NIST Special Publication 260-140. recertification of the absorbance values of these filters is
The certified absorbances should be traceable to the regular required (due to expiration or improper storage or handling)
transmittance scale maintained by a national metrology insti- they should be returned to the certifying laboratory for cleaning
tute (NMI). and recertification.
9.1.1 If the blank-corrected absorbances (Acorr) of the stan- 9.2.2 Procedure:
dards are outside the acceptable range, then corrective service 9.2.2.1 Examine the glass filters for surface contamination
must be performed on the instrument by a qualified service and clean with a bulb-type of air puffer if necessary. Any other
person. If the user performs this service, the manufacturer’s attempt to clean the filters invalidates the certification. Measure
recommended procedure should be followed carefully. the temperature of the sample compartment as described in
9.1.2 An acceptable absorbance range for each standard for Section 7.
any instrument must be determined based on the instrument
9.2.2.2 Determine the absorbance blank (air versus air
manufacturer’s specifications and on the analytical demands of
absorbance value) at the indicated wavelengths. Record these
the end-use application of the instrument. As a guide to the
measurements. If large (>0.001A) blank values are observed,
acceptability of photometric accuracy data a tolerance of
use these to blank-correct measured apparent absorbances by
61.0 % relative (0.2 # A # 2.0) is employed in this practice.
subtraction. Measure the apparent absorbance of each filter at
The user is encouraged to establish tolerance limits more
each wavelength versus air. Each filter should be oriented in
appropriate to the application in question, and use the tables of
the same manner in the sample holder. If a corrected absor-
this practice as templates for custom tables that reflect the
bance reading is outside the acceptable absorbance range,
appropriate tolerances.
repeat the procedure with a longer integration time and smaller
9.1.3 Rigorous treatment of the construction and use of an
spectral slit width, if possible, to verify the nonconformity.
absorbance correction curve for high accuracy work is beyond
9.2.3 Report the visible region validation data in the manner
the scope of this practice.
of Table 2, constructed for a set of three filters of the nominal
9.1.4 Studies by NIST have indicated that solutions of
absorbances of NIST SRM 930.
acidic potassium dichromate are stable for at least six months
when prepared in the manner described in 9.3.2.1 and stored in 9.3 Ultraviolet Region—The absorbance scale in the ultra-
the dark in well-stoppered 1-L volumetric flasks. Neutral violet region is tested using acidic solutions of potassium
density glass filters are certified by different sources for periods dichromate (available from NIST as SRM 935a). The wave-
of from two to five years, with appropriately adjusted uncer- lengths of interest are:
tainties. 235 nm
9.2 Visible Region—The absorbance scale in the visible 257 nm
region is tested using filters of a proprietary neutral glass. The 313 nm
construction and certification of such filters is described in 350 nm
some detail in NIST Special Publications SP 260-116 and SP NOTE 1—Acidic potassium dichromate solutions specifically prepared
260-140. Traceability of the certified absorbance values to the for spectrophotometric validation are also available commercially in

4
 E 925
TABLE 2 UV-VIS Spectrophotometers Absorbance Calibration—Visible Region
Instrument
Date
Temperature
Analyst

Wavelength ConformanceD
Filter No. AnomA AcertA AcorrA BiasB ToleranceC
(nm) Does Does Not
440.0 0.5 0.005
0.7 0.007
1.0 0.010

465.0 0.5 0.005

0.7 0.007
1.0 0.010

546.1 0.5 0.005

0.7 0.007
1.0 0.010

590.0 0.5 0.005

0.7 0.007
1.0 0.010

635.0 0.5 0.005

0.7 0.007
1.0 0.010

A
Anom = nominal absorbance; Acert = certified absorbance; Acorr = measured absorbance, blank corrected as necessary.
B
Bias = Acorr − Acert.
C
Tolerance taken for example as 1 % of the nominal. User to assign as appropriate for each application.
D
Measurement conforms for |Bias| # Tolerance; measurement does not conform for |Bias| > Tolerance.

sealed ampoules and cuvettes. Portions of the procedure below, for the Measure the temperature of the sample compartment as de-
powder form, will not be required for these forms. Certified values and scribed in Section 7.
expiration dates that accompany such preparations should be observed.
9.3.2.3 Determine the apparent absorbance blank at the
9.3.1 The precautionary notes stated in the certificate and indicated wavelengths using solvent in each cuvette. Record
the material safety data sheet (MSDS) for SRM 935a should be these measurements. If large (>0.01A) blank values are ob-
observed. These documents are available from the NIST served, re-cleaning the cuvettes may be necessary. Measure the
internet site at www.nist.gov under the Standard Reference apparent absorbance of each Absorbance Standard Sample
Materials Program online catalog. Calibration Solution of potassium dichromate in the sample
9.3.2 Procedure: cuvette at each wavelength and record. Rinse the cuvettes
9.3.2.1 Prepare the absorbance standard solutions of potas-
several times with the solutions to be measured before they are
sium dichromate by transferring 200.0 6 0.3, 300.0 6 0.3,
placed in the sample compartment and maintain the same
400.0 6 0.3, and 500.0 6 0.3 mg of the powder to four
orientation of a cuvette throughout the procedure. If a corrected
separate 100 mL volumetric flasks and dilute to volume with
apparent absorbance value (Acorr) of an Absorbance Standard
distilled water (Absorbance Standard Stock Solutions). Stopper
Sample Calibration is outside the acceptable range, repeat the
the solutions and mix well. Dilute these solutions by pipetting
20.0 mL of each solution separately to four 1-L volumetric reading with a longer integration time and smaller spectral slit
flasks, adding 1 mL of 1M HClO4 (8.6 mL of 70 % HClO4/100 width, if possible. If the absorbance readings at all wavelengths
mL H2O) and diluting to volume with distilled water (Absor- for a solution are unacceptable, prepare a fresh solution at the
bance Standard Sample Calibration Solutions). These final concentration of interest and repeat the absorbance measure-
calibration solutions contain 40, 60, 80, and 100 mg of ments. If non-conformities are verified, corrective service must
potassium dichromate per litre of solution, respectively. Pre- be performed by a qualified service person. If the user performs
pare a blank solution by diluting 1 mL of 1 M HClO4 to one L this service, the manufacturer’s recommended procedure
with the same distilled water. Stopper the solutions and mix should be followed carefully.
well. 9.3.3 Report the ultraviolet region calibration data in the
9.3.2.2 Clean and match the 1-cm solution cells (cuvettes). manner of Table 3.

5
 E 925
TABLE 3 UV-VIS Spectrophotometer Absorbance Calibration—Ultraviolet Region (Potassium Dichromate)
Instrument
Date
Temperature
Analyst

Wavelength Solution ConformanceE

AcertA AmeasB AblankB AcorrB BiasC ToleranceD
(nm) (mg/L) Does Does Not
235.0 40 0.492 0.005
60 0.741 0.007
80 0.991 0.010
100 1.243 0.012

257.0 40 0.573 0.006

60 0.862 0.009
80 1.154 0.012
100 1.449 0.014

313.0 40 0.192 0.002

60 0.289 0.003
80 0.386 0.004
100 0.483 0.005

350.0 40 0.427 0.004

60 0.645 0.006
80 0.860 0.009
100 1.071 0.011

A
Acert = certified absorbance, computed from SRM 935a certified specific absorbance values for the given solution and a 10-mm pathlength.
B
Ameas = measured solution absorbance; Ablank = measured blank absorbance; Acorr = Ameas − Ablank.
C
Bias = Acorr − Acert.
D
Tolerance computed for illustrative purposes as 1 % of the certified absorbance. User may substitute appropriate tolerances.
E
Corrected absorbance conforms when |Bias| # Tolerance.

10. Documentation of Data 11. Keywords

10.1 Spectral charts and tables should be retained for 11.1 absorbance; molecular spectroscopy; reference materi-
reference. als; spectrophotometers; UV/visible; wavelength

REFERENCES

(1) McNeirney, J., and Slavin, W., Applied Optics, Vol 1, 1962, p. 365. (2) Keegan, H. J, Schleter, J. C., and Weidner, V. R., J. Opt. Soc. Am., Vol
51, 1961, p. 1470.

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